Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.10.2 (focal adhesion kinase)
 44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The use of consensus values in external quality assessment schemes (EQAS) involves several problems and should preferably be replaced with target values obtained by methods of high metrological level. However, such values are difficult to obtain. In the present study we transferred values from the NIST (former NBS) certified reference serum SRM 909 to lyophilized and frozen test sera for various inorganic components using flame absorption or flame emission spectrometry. Enzyme values were assigned by laboratories of members of the former Scandinavian Enzyme Committee. The assignment was based on 2-4 determinations each day through 3 days of experiment. A total of 10 laboratories participated in the work. The results were utilized in a Danish EQAS. One practical concern is the fairly long time (9 months) which was needed for production, collection and compiling all data. To get an impression of how much dry chemistry analysers, e.g, could influence consensus values a Kodak Ektachem 700 XR was studied using lyophilized and frozen sera. The results are reported in the annex. On NIST SRM 909 the values found for sodium(I) were 6% too high even though the findings on frozen human sera were accurate. For aspartate aminotransferase a result three times the target values was found on a human lyophilized serum, while the values on the frozen sera only were slightly too high.
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PMID:A programme for assigning target values for external quality assessment schemes in countries with no authorized reference laboratories. Annex. Experiences with deviating results on Ektachem 700 XR. 846 50

There is now considerable evidence that nitric oxide (NO) is an important neuroregulatory agent, but there has been very little investigation of the possible role of NO in neuroendocrine mechanisms. We have previously shown that acute rat hypothalamic explants can be used to study the regulation of hypothalamic neuropeptide release, and we have now utilised this experimental approach to investigate the putative involvement of NO in the control of the principal corticotropin-releasing hormone, CRH. We studied the direct effects of the NO precursor L-arginine (L-ARG), as well as the NO donors molsidomine and sodium nitroprusside, on both the basal and stimulated release of CRH; the stimuli used were non-specific depolarisation with potassium chloride (KCl) and the specific cytokine, interleukin-1 beta (IL-1 beta; 100 U/ml). L-ARG was tested in each experimental condition with and without contemporaneous addition of its competitive antagonist NG-monomethyl-L-arginine (L-NMMA). IL-1 beta-induced CRH release was also investigated in the presence of D-arginine (D-ARG), which is not active as a precursor to NO, and ferrous hemoglobin (Hb), a substance which is a potent inactivator of NO. None of the NO precursors (L-ARG, molsidomine, sodium nitroprusside) or antagonists (L-NMMA or Hb) was able to affect basal CRH release. However, L-ARG 10 and 100 microM were found to significantly inhibit the release of CRH induced by 40 mM KCl; CRH fell to 45% of its stimulated level at the higher dose of L-ARG. This effect was attenuated in the presence of L-NMMA at a ten-fold higher dose.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nitric oxide modulates the release of corticotropin-releasing hormone from the rat hypothalamus in vitro. 848 68

Mean values of extracellular pH (pHe) in tumours tend to be about 0.5 pH units lower than in normal tissues, whereas values of intracellular pH (pHi) in tumours and normal tissues are similar. Previous studies have shown that drugs that acidify cells at lower pHe such as nigericin, used alone or with agents that inhibit the regulation of pHi, have toxicity to cultured cells at pHe < 6.5 in short-term exposure; these agents also lead to modest anti-tumour effects in mice when given acutely. To evaluate the long-term effects of these drugs at levels of pHe that might occur commonly in tumours, we exposed cells for up to 72h at pHe 6.8 or 7.2 in vitro. Nigericin (0.033 microM) caused time-dependent cell killing of murine KHT and EMT-6 cells at pHe 6.8 (but not at pHe 7.2) with a surviving fraction approximately 5 x 10(-3) after 72 h exposure. Cell killing was increased in the presence of 4,4-diisothiocyanstilbene 2,2-disulphonic acid (DIDS), an inhibitor of Na+-dependent HCO3-/CI- exchange, and to a lesser extent in the presence of 5-(N-ethyl-N-isopropyl) amiloride (EIPA), an inhibitor of Na+/H+ exchange. Cell killing was exquisitely sensitive to the level of pHe. Osmotic pumps were used to obtain a 72 h continuous infusion of nigericin in mice; this led to dose-dependent killing of cells in KHT tumours with surviving fraction of approximately 0.1 at maximum tolerated doses. Hydralazine, which may cause tumour hypoxia and lower pHi as well as pHe, caused cytotoxity when given alone by chronic infusion, and enhanced the cytotoxicity due to nigericin. The addition of DIDS and/or EIPA (using two pumps) further enhanced anti-tumour toxicity, with a surviving fraction of approximately 0.002 at tolerated doses of the four drugs used to treat KHT tumours. The experiments demonstrate the activity of drugs that inhibit the regulation of pHi against murine tumours when delivered by chronic infusion.
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PMID:The chronic administration of drugs that inhibit the regulation of intracellular pH: in vitro and anti-tumour effects. 864 75

Detachment of basal keratinocytes from basement membrane signals a differentiation cascade. Two integrin receptors alpha6beta4 and alpha3beta1 mediate adhesion to laminin 5 (epiligrin), a major extracellular matrix protein in the basement membrane of epidermis. By establishing a low temperature adhesion system at 4 degrees C, we were able to examine the exclusive role of alpha6beta4 in adhesion of human foreskin keratinocyte (HFK) and the colon carcinoma cell LS123. We identified a novel 80-kD membrane-associated protein (p80) that is tyrosine phosphorylated in response to dissociation of alpha6beta4 from laminin 5. The specificity of p80 phosphorylation for laminin 5 and alpha6beta4 was illustrated by the lack of regulation of p80 phosphorylation on collagen, fibronectin, or poly-L-lysine surfaces. We showed that blocking of alpha3beta1 function using inhibitory mAbs, low temperature, or cytochalasin D diminished tyrosine phosphorylation of focal adhesion kinase but not p80 phosphorylation. Therefore, under our assay conditions, p80 phosphorylation is regulated by alpha6beta4, while motility via alpha3beta1 causes phosphorylation of focal adhesion kinase. Consistent with a linkage between p80 dephosphorylation and alpha6beta4 anchorage to laminin 5, we found that phosphatase inhibitor sodium vanadate, which blocked the p80 dephosphorylation, prevented the alpha6beta4-dependent cell anchorage to laminin 5 at 4degreesC. In contrast, adhesion at 37 degrees C via alpha3beta1 was unaffected. Furthermore, by in vitro kinase assay, we identified a kinase activity for p80 phosphorylation in suspended HFKs but not in attached cells. The kinase activity, alpha6beta4, and its associated adhesion structure stable anchoring contacts were all cofractionated in the Triton-insoluble cell fraction that lacks alpha3beta1. Thus, regulation of p80 phosphorylation, through the activities of p80 kinase and phosphatase, correlates with alpha6beta4-SAC anchorage to laminin 5 at 4 degrees C in epithelial cells of the skin and intestine. Transmembrane signaling through p80 is an early tyrosine phosphorylation event responsive to and possibly required for anchorage to laminin 5 by HFK and LS123 epithelial cells.
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PMID:Anchorage mediated by integrin alpha6beta4 to laminin 5 (epiligrin) regulates tyrosine phosphorylation of a membrane-associated 80-kD protein. 864 1

A procedure for renaturing and detecting the activity of protein tyrosine phosphatases (PTPases) after sodium dodecyl sulfate (SDS)-gel electrophoresis with greatly improved sensitivity and resolution is described. Epidermal growth factor receptor-kinase, c-src kinase, and focal adhesion kinase were phosphorylated on tyrosine with 32PO4 and incorporated into gels prior to electrophoresis. These proteins are dephosphorylated when cellular proteins are electrophoresed and the separated PTPases are renatured in the gel by removing SDS with extensive washing. With whole cell lysates, at least eight separate bands of decreased radioactivity corresponding to PTPase activity with molecular weights between 110 and 34 kDa are seen in autoradiographs of the dried gels. PTPases detected are similar with different cell types and with the three 32P-labeled protein substrates, although they are different in cytosolic and membrane-associated fractions. A PTPase detected above 200 kDa in wheat germ agglutinin eluates from solubilized cells suggests that some receptor PTPases can be renatured. While microgram levels of recombinant PTP-1C are required for detection, nanogram levels of recombinant PTP-1B are easily detected. Assaying the activity of renatured PTPases after they have been separated by molecular weight in SDS gel electrophoresis provides a simple and rapid means of determining the activity of individual PTPases in cell fractions.
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PMID:Activity and molecular weight of protein tyrosine phosphatases in cell lysates determined by renaturation after gel electrophoresis. 866 May 68

Because of the well established role that tyrosine phosphorylation (tyr phos) plays in growth factor signalling and regulating cell growth, we hypothesized that cardiac hypertrophy might be associated with altered tyr phos of certain cellular proteins in the heart. Furthermore, we hypothesized that angiotensin II (ang II), a putative growth factor for cardiac cells, might be useful as a probe to highlight any differences in intracellular signalling between normal and hypertrophied hearts. The heart and, for comparison, skeletal muscle, from Dahl S rats, which are predisposed to cardiac hypertrophy, and Dahl R rats, which are not, were examined. Antiphosphotyrosine immunoprecipitation and immunoblotting of heart cell extracts revealed the presence of a constitutively tyr phos 120 kDa cytosolic protein. Hearts from Dahl R rats on a high salt diet displayed a smaller amount of constitutive tyr phos of this protein. In the hearts of both Dahl R and S rats maintained on low salt diets there was little evidence of constitutive tyr phos of this protein. Ang II induced tyr phos of this protein in Dahl S rats on a low salt diet and Dahl R rats on a high salt diet, both of which show mild cardiac hypertrophy. In contrast, the markedly hypertrophied ventricle showed a minimal response to Ang II. Thus the severity of cardiac hypertrophy correlated directly with the tyr phos level of this protein. In an attempt to identify this protein, immunoblotting was carried out with antibodies to the signal transducing proteins rasGAP, JAK2 iNOS, p125FAK, and the Src substrate, pp120, but all proved negative. Ang II also stimulated an increase in tyr phos of proteins with apparent molecular masses of 42, 55, and 69 to 85 kDa in hearts from Dahl S rats on high salt diet. By comparison, there was no 120 kDa tyr phos protein in skeletal muscle even in response to Ang II. Silver stained sodium dodecyl sulfate gels demonstrated that this 120 kDa tyr phos protein is present in substantial amounts in the ventricles of rats fed high salt diets. Thus cardiac hypertrophy is characterized by an abundant 120 kDa cytosolic tyr phos protein, which is apparent with Ang II stimulation in milder degrees of cardiac hypertrophy, and is most likely an as yet uncharacterized protein.
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PMID:Cardiac hypertrophy in the Dahl rat is associated with increased tyrosine phosphorylation of several cytosolic proteins, including a 120 kDa protein. 869 21

The study was conducted to investigate the thermoregulation of young children compared to that of adults. A group of 19 children (ages 9 months-4.5 years), with only 3 children aged 3 years or above, and 16 adults first rested in a thermoneutral room (air temperature 25 degrees C relative humidity 50%, air velocity 0.2 m.s(-1)). They were then exposed to a hot room (air temperature 35 degrees C, relative humidity 70%, air velocity 0.3 m.s(-1)) next door for 30 min, and then returned to the thermoneutral room where they stayed for a further 30 min. The rectal temperature (Tre), skin temperatures (Tsk) at seven sites, heart rate (HR), total sweat rate (Msw,t), local sweat rate (Msw,l) and the Na+ concentration of the sweat were measured. There was no significant difference in Tre between the children and their mothers in the rest phase. However, the Tre of the children increased as soon as they entered the hot room and was significantly higher than during the control period, and than that of the mothers during heat exposure. Mean Tsk, forehead, abdomen and instep Tsk were significantly higher in the children during both the thermoneutral and heat exposure. The Msw,t was significantly higher and Na+ concentrations in the sweat on the back and upperarm were significantly lower for the children during the heat exposure. They had a greater body surface area-to-mass ratio than the mothers by 64%, which indicated that they had advantages for thermal regulation. However, the sweating and Tsk responses of the children were not enough to prevent a rise in body temperature. These results would suggest that the young children had the disadvantage of heating up easily due to their smaller body sizes and there may be maturation-related differences in thermoregulation during the heat exposure between young children and mothers.
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PMID:Thermoregulation during heat exposure of young children compared to their mothers. 878 64

We studied the influence of dietary L-arginine (L-ARG) supplementation on forearm resistance arteries in vivo and the effect of exogenous addition of L-ARG to subcutaneous arteries isolated from gluteal biopsies. Twenty-six healthy males were recruited, and 16 were randomly allocated in a double-blind protocol to receive either oral L-ARG 20 g/day or placebo for 28 days. We examined responses to acetylcholine (ACh), sodium nitroprusside (SNP) and NG-monomethyl-L-arginine (L-NMMA) on forearm resistance arteries using venous occlusion plethysmography performed before and after supplementation of L-ARG (or placebo). L-ARG 20 g/day had no effect on plasma L-ARG levels (% mol based on total amino acid pool; before vs. after L-ARG 3.43 +/- 0.31 vs. 3.76 +/- 0.05), weekly blood pressure (BP) measurements, or plasma biochemical analysis of liver function enzymes, urea, or electrolyte levels. On the other hand, analysis of the major amino acids in plasma showed a significant difference in profile after L-ARG, but not placebo supplementation (Mann Whitney U test, p < 0.05), indicating a domino effect of chronic oral L-ARG supplementation on other amino acids. This may result from a change in appetite and thus protein intake after L-ARG supplementation. At the dose given, neither L-ARG nor placebo had any effect on forearm blood flow (FBF) responses to ACh (area under the dose-response curve, before vs. after L-ARG 1,763 +/- 260.1 vs. 1,862.8 +/- 163.6 U, Student's paired t test; p > 0.05), SNP, or L-NMMA. Gluteal skin biopsies were performed on 10 different untreated subjects. Subcutaneous arteries were isolated and mounted as ring preparations in isometric small vessel myographs. Full concentration-response curves to norepinephrine (NE), ACh, substance P, and a single response to SNP (10 microM) were obtained with and without addition of either L- or D-ARG 10 microM. Both L-ARG [-log EC50 (M) before vs. after arginine 7.12 +/- 0.15 vs. 6.66 +/- 0.16, Student's paired t test, p < 0.005] and D-ARG [-log EC50 (M) before vs. after arginine 7.36 +/- 0.17 vs. 6.85 +/- 0.18; Student's paired t test, p < 0.05] significantly antagonized responses to NE in subcutaneous arteries isolated from healthy humans. With the exception of a subset of vessels in which some endothelial dysfunction was observed, neither of the isomers of arginine had any effect on the responses to ACh, substance P, or SNP. In the subset vessels already described (n = 5), in which responses to ACh were < 90% maximal dilatation, L- but not D-ARG significantly increased the potency to ACh [-log EC50 (M) before vs. after L-ARG 7.42 +/- 0.20 vs. 8.27 +/- 0.28. Student's paired t test, p < 0.05]. We conclude that oral supplementation with L-ARG 20 g/day for 28 days does not affect endothelial function in normal healthy adults, possibly because the dose given in the current study was inadequate or because chronic oral administration leads to dissipation of arginine to other pathways, as evidenced by the change in total amino acid profile but not L-ARG plasma concentration, or because L-ARG cannot improve normal endothelium-mediated vasodilatation. These concepts are supported by our findings that responses to ACh and substance P were not altered by L-ARG in subcutaneous arteries isolated from healthy subjects.
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PMID:Effects of in vivo and in vitro L-arginine supplementation on healthy human vessels. 879 50

In whole-cell recordings from CA1 neurons, net outward currents (at ca. -20 mV, from VH ca. -50 mV) were 40-50% depressed by sodium nitroprusside (100-500 microM) or L-arginine (L-ARG; 50-200 microM), but not by D-arginine (100 microM). The NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME; 200 microM) restored the L-ARG-depressed current to ca. 80% of control. In naive cells, L-NAME increased outward currents by 45 +/- 12.6%; the enhanced currents were then reduced by adding L-ARG (200-400 microM). The NO-sensitive current is Ca-dependent, because L-NAME and L-ARG were ineffective in Mn/low Ca medium or when electrodes contained 2.2 mM EGTA. Since high voltage-activated Ca-currents were unaltered by L-NAME, we conclude that NO tonically enhances excitability in slices by depressing a voltage- and calcium-dependent (IK(Ca)-type) outward current.
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PMID:Nitric oxide tonically depresses a voltage- and Ca-dependent outward current in hippocampal slices. 883 Mar 13

The sodium-hydrogen exchanger (NHE) has been implicated in bone resorption by osteoclasts. We have studied expression of NHE-1, an isoform of the NHE, in chicken bone marrow mononuclear phagocyte precursors during differentiation into the osteoclast phenotype in culture. A monoclonal anti-body raised against the carboxy-terminus of NHE-1 detected the presence of a 100 kDa protein (similar to the mammalian form of NHE-1) in the osteoclasts. Laser scanning confocal microscopy revealed association with the alpha(v)beta(3) integrin and focal adhesion kinase (pp(125)FAK) at the basolateral membrane (BLM) of the osteoclast in addition to a more generalized cellular distribution. A fragment of avian NHE-1 cDNA was obtained by polymerase chain reaction cloning, and it was used to characterize expression of NHE-1 transcripts in cultured chicken osteoclast precursors. The avian NHE-1 message was a 3.9 kB band on Northern analysis, which differed from the mammalian message. Retinoic acid (RA) elicited an increase in the steady-state intracellular pH (pH(1)) from 6.87 to 7.10 in the absence of bicarbonate and was inhibited by ethylisopropylamiloride, an inhibitor of Na-H exchange. Using ribonuclease protection assays, we found that NHE-1 transcripts are induced as cells differentiate in vitro and in response to 13-cis-RA. Western blot analysis indicated that protein levels also increased in response to 13-cis-RA. Our results demonstrate expression of NHE-1 in avian osteoclasts with a complex cellular distribution in culture, and NHE-1 expression is induced as cells differentiate into mature osteoclasts in response to 13-cis-RA.
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PMID:Cellular distribution and regulation of NHE-1 isoform of the NA-H exchanger in the avian osteoclast. 883 1


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